The identification of early genetic changes in tumorigenesis is a primary focus in molecular cancer research. Characterization of the nature and pattern of cancer-associated genetic alterations will allow for early detection, diagnosis and treatment of cancer. Such genetic alterations in vertebrates fall generally into one of three categories: gain or loss of genetic material; mutation of genetic material; or methylation at cytosine residues in CpG dinucleotides within “CpG islands.” Among these, DNA methylation is uniquely reversible, and changes in methylation state are known to affect gene expression (e.g., transcriptional initiation of genes where CpG islands located at or near the promoter region) or genomic stability.
Methylation of CpG dinucleotides within CpG islands. DNA, in higher order eukaryotic organisms, is methylated only at cytosine residues located 5′ to guanosine residues in CpG dinucleotides. This covalent modification of the C-5 position of the cytosine base by the enzyme DNA (cytosine-5)-methyltransferase results in the formation of 5-methylcytosine (5-mCyt), and gives this base unique properties (e.g., susceptibility to undergo spontaneous deamination). This enzymatic conversion is the only epigenetic modification of DNA known to exist in vertebrates, and is essential for normal embryonic development (Bird, A. P., Cell 70:5-8, 1992; Laird & Jaenisch, Human Molecular Genetics 3:1487-1495, 1994; Li et al., Cell 69:915-926, 1992).
The presence of 5-mCyt at CpG dinucleotides has resulted in the 5-fold depletion of this sequence in the genome during the course of vertebrate evolution (Schroderet & Gartler, Proc. Nat. Acad. Sci. USA 89:957-961, 1992), presumably due to spontaneous deamination of 5-mCyt to Thymidine. Certain areas of the genome, however, do not show such depletion, and are referred to as “CpG islands” (Bird, A. P., Nature 321:209-213, 1986; Gardiner-Garden & Frommer, J. Mol. Biol. 196:261-282, 1987). These CpG islands comprise only approximately 1% of the vertebrate genome, yet account for about 15% of the total number of genomic CpG dinucleotides (Antequera & Bird, Proc. Nat. Acad. Sci. USA 90:11995-11999, 1993). CpG islands contain the expected (i.e., the non-evolutionarily depleted) frequency of CpGs (with an Observed/Expected Ratio1>0.6), are GC-rich (with a GC Content2>0.5) and are typically between about 0.2 to about 1 kb in length. 1Calculated as: [number of CpG sites/(number of C bases×number of G bases)]×band length for each fragment.2 Calculated as: (number of C bases+number of G bases)/band length for each fragment.
Methylation within CpG islands affects gene expression. CpG islands are located upstream of many housekeeping and tissue-specific genes, but may also extend into gene coding regions (Cross & Bird, Current Opinions in Genetics and Development 5:309-314, 1995; Larsen et al., Genomics 13:1095-1107, 1992). The methylation of cytosines within CpG islands in somatic tissues is believed to affect gene expression. Methylation has been inversely correlated with gene activity and may lead to decreased gene expression by a variety of mechanisms including inhibition of transcription initiation (Bird, A. P., Nature 321:209-213, 1986; Delgado et al., EMBO Journal 17:2426-2435, 1998), disruption of local chromatin structure (Counts & Goodman, Molecular Carcinogenesis 11:185-188, 1994; Antequera et al., Cell 62:503-514, 1990), and recruitment of proteins that interact specifically with methylated sequences and thereby directly or indirectly prevent transcription factor binding (Bird, A. P., Cell 70:5-8, 1992; Counts & Goodman, Molecular Carcinogenesis 11:185-188, 1994; Cedar, H., Cell 53:3-4, 1988). Many studies have demonstrated the effect of methylation of CpG islands on gene expression (e.g., the CDKN2A/p16 gene; Gonzalez-Zulueta et al., Cancer Research 55:4531-4535, 1995), but most CpG islands on autosomal genes remain unmethylated in the germline, and methylation of these islands is usually independent of gene expression. Tissue-specific genes are typically unmethylated in the respective target organs but are methylated in the germline and in non-expressing adult tissues, while CpG islands of constitutively expressed housekeeping genes are normally unmethylated in the germline and in somatic tissues.
Methylation within CpG islands affects the expression of genes involved in cancer. Data from a group of studies show the presence of altered methylation in cancer cells relative to non-cancerous cells. These studies show not only alteration of the overall genomic levels of DNA methylation, but also changes in the distribution of methyl groups. For example, abnormal methylation of CpG islands that are associated with tumor suppressor genes or oncogenes within a cell may cause altered gene expression. Such altered gene expression may provide a population of cells with a selective growth advantage and thereby result in selection of these cells to the detriment of the organism (i.e., cancer).
Insufficient correlative data. Unfortunately, the mere knowledge of the basic existence of altered methylation of CpG dinucleotides within CpG islands of cancer cells relative to normal cells, or of the fact that in particular instances such methylation changes result in altered gene expression (or chromatin structure or stability), is inadequate to allow for effective diagnostic, prognostic and therapeutic application of this knowledge. This is because only a limited number of CpG islands have been characterized, and thus there is insufficient knowledge, as to which particular CpG islands, among many, are actually involved in, or show significant correlation with cancer or the etiology thereof. Moreover, complex methylation patterns, involving a plurality of methylation-altered DNA sequences, including those that may have the sequence composition to qualify as CpG islands, may exist in particular cancers.
Therefore there is a need in the art to identify and characterize specific methylation altered DNA sequences, and to correlate them with cancer to allow for their diagnostic, prognostic and therapeutic application.